JP2014523906A - Oral treatment of inflammatory bowel disease - Google Patents

Abstract

The present invention relates to the treatment of inflammatory bowel disease by simultaneous or sequential parenteral and oral administration of mammalian beta-defensins.
[Selection] Figure 1

Description

  The present invention relates to the treatment of inflammatory bowel disease by simultaneous or sequential parenteral and oral administration of mammalian beta-defensins.

  Human defensin

  Among the many elements, particularly important components for innate immunity are antimicrobial peptides (AMP) that can individually kill a wide range of bacteria, viruses, and fungi, while exhibiting high selectivity individually. The biological significance of AMP is even more pronounced because it is ubiquitously distributed in nature and is probably produced in all multicellular organisms. In humans, the major AMP is defensin. Human defensins are small cationic peptides that can be classified into α- and β-defensins based on the topology of three intracellular cysteine disulfide bonds. α-Defensins can be further subdivided into those first isolated from neutrophil granules (HNP1-4) and those expressed by Paneth cells in the crypts of the small intestine (HD5 and HD6). it can. β-defensins are mainly produced by epithelial cells of various tissues and organs, including skin, trachea, gastrointestinal tract, urogenital system, kidney, pancreas, and mammary gland. Among the β-defensin families, hBD1 to 3 have the most advanced characterization. However, using various bioinformatics tools, the human genome has been annotated with about 40 open reading frames encoding putative β-defensin homologues. Some human defensins are constitutively produced while others are induced by inflammatory cytokines or exogenous microbial products.

  In addition to its direct antibacterial activity, human defensin has gradually become clear to have a wide range of immunomodulatory / alternative properties. These include induction of various chemokines and cytokines, chemotaxis and apoptotic activity, induction of prostaglandin, histamine and leukotriene release, complement inhibition, and dendritic cell maturation by Toll-like receptor signaling. Stimulation and stimulation of pathogen clearance by neutrophils are included. In addition, human defensins play a role in wound healing, epithelial and fibroblast proliferation, angiogenesis, and angiogenesis.

  There is increasing evidence that human defensin plays an important role in many infectious and inflammatory diseases. Overexpression of human defensin is often observed in inflamed and / or infected skin, which is likely due to local induction by microbial components or endogenous inflammatory cytokines. In psoriasis, hBD2 and hBD3 become excessive, and significant upregulation of hBD2 has been observed in the lesion epithelium of patients with acne vulgaris or superficial folliculitis. On the other hand, down-regulation of hBD2 and hBD3 is associated with atopic dermatitis. Ileal Crohn's disease is associated with a lack of expression of HD5 and HD6, and in Crohn's disease the expression of hBD2-4 is down-regulated in the colon.

  Inflammatory bowel disease

  Inflammatory bowel disease (IBD) is defined as chronic recurrent bowel disease of unknown cause. IBD refers to two types of diseases, Crohn's disease and ulcerative colitis (UC). Both diseases appear to result from uninhibited activation of the inflammatory response in the intestine. This inflammatory cascade is thought to continue through the action of inflammatory cytokines and selective activation of lymphocyte subsets. In patients with IBD, lining ulcers and inflammation inside the intestine lead to symptoms of abdominal pain, diarrhea, and rectal bleeding. Although ulcerative colitis occurs in the large intestine, in Crohn's disease, the disease can be involved in the entire gastrointestinal tract as well as the small and large intestine. For most patients, IBD is a chronic disease with symptoms that last for months to years. Although most common in young adults, it can occur at any age. Although it is seen all over the world, it is most common in developed countries such as the United States, the United Kingdom, and Northern Europe. It is especially common in Jewish people, and there is a difference in morbidity among races. Recent studies have identified specific genes, including ATG16L1, IL23R, IRGM, and NOD2, that affect the risk of developing Crohn's disease. As many as 30 human genes have been identified that promote susceptibility to ulcerative colitis.

  Clinical symptoms of IBD are intermittent rectal bleeding, convulsive abdominal pain, weight loss, and diarrhea. The diagnosis of IBD is based on clinical symptoms, the use of barium enema, but direct visualization (sigmoidoscopy or colonoscopy) is the most accurate test. Prolonged IBD is a risk factor for colon cancer, and treatment of IBD may include medication and surgery.

  Some UC patients have disease only in the rectum (proctitis). Other UC patients have disease confined to the rectum and adjacent left colon (recto-sigmoiditis). Still other patients have UC throughout the colon (generic IBD). Symptoms of UC are generally severe in a wider range of diseases (when the part of the colon involved in the disease is large).

  The prognosis for patients with disease limited to the rectum (proctitis) or UC limited to the left colonic end (rectosigmoiditis) is better than patients with whole colon UC. Short periods of regular treatment with oral or enema can be sufficient. In patients with a wider range of diseases, blood loss from the inflamed intestine may lead to anemia and may require treatment with iron supplements or blood transfusions. Rarely, when the inflammation is very severe, the colon can expand acutely and become large in size. This condition is called toxic megacolon. Patients with toxic megacolon are extremely exacerbated by fever, abdominal pain and swelling, dehydration, and malnutrition. If medication does not improve the patient quickly, it is usually necessary to prevent colon rupture by surgery.

  Crohn's disease can occur in all areas of the gastrointestinal tract. With this disease, intestinal obstruction due to inflammation and fibrosis occurs in many patients. Granulomas and fistula formation are common complications of Crohn's disease. Results of disease progression include parenteral nutrition, surgery, and colostomy.

  IBD can be treated with drugs. The most commonly used drugs for treating IBD are anti-inflammatory drugs such as salicylate. Salicylate preparations have been effective in treating mild to moderate diseases. Furthermore, when medication is performed over a long period of time, the frequency of relapse of the disease can be reduced. Examples of salicylates include sulfasalazine, asalphidin, olsalazine, and mesalamine. All of these drugs are administered at high doses orally for maximum therapeutic effect. These drugs are not without side effects. Asalphidin can cause upset stomach when administered at high doses, and in rare cases, mild nephritis has been reported in some salicylate preparations.

  Corticosteroids are more powerful and immediate than salicylate in the treatment of IBD, but the use of corticosteroids is limited to patients with high severity of the disease because of the potential for serious side effects. The side effects of corticosteroids are usually caused by long-term use. Side effects include bone and skin thinning, increased susceptibility to infection, diabetes, hypertension, glaucoma, muscle wasting / debilitation, round face, psychiatric disorders, and, rarely, hip joint destruction.

  In IBD patients who do not respond to salicylate or corticosteroids, drugs that suppress the immune system are used. Examples of immunosuppressive agents include azathioprine and 6-mercaptopurine. The immunosuppressive agent used in this situation is useful for the suppression of IBD and allows a gradual reduction or elimination of corticosteroids. However, immunosuppressive agents make patients less immune and more susceptible to many other diseases.

  A well-known model for IBD research is the DSS colitis mouse model, which is described in Non-Patent Document 1 and Non-Patent Document 2.

Kawada et al. “Insights from advances in research of chemically induced experimental models of human inflammatory bowel disease”, World J. Gastroenterol., Vol. 13 (42), pp. 5581-5593 (2007) Wirtz and Neurath “Mouse models of inflammatory bowel disease”, Advanced Drug Delivery Reviews, Vol. 59 (11), 1073-1083 (2007)

  Treatment of inflammatory bowel disease (IBD) has traditionally been achieved by administration of aminosalicylate, corticosteroids, thiopurine, methotrexate, and anti-tumor necrosis factor agents. Human beta defensin has long been thought to play a role in the development and / or treatment of IBD.

  Clearly there is a great need for drugs that can prevent and treat IBD.

  WO2007 / 081486 discloses the use of several human defensins in the treatment of inflammatory bowel disease. The inventor found that defensin administered orally for Crohn's disease reduces the number of invading bacteria and restores normal epithelial barrier function in a dosage form that can be released at the appropriate location in the intestinal lumen. It has been shown to reduce the severity of inflammatory diseases.

  According to WO2007 / 081486, the function of defensin is to kill bacteria directly in the lumen as a target and prevent entry into epithelial tissues. That is, the function of defensin is purely similar to that of anti-infective compounds.

  The present invention demonstrates anti-inflammatory activity after a 10-day treatment period of orally administered hBD2 in a mouse-induced DSS colitis model.

  These data show the beneficial therapeutic effects of IBD associated with simultaneous oral and parenteral administration of mammalian beta-defensin.

  In one aspect, the invention relates to a method of administering mammalian beta defensin to an individual suffering from inflammatory bowel disease, comprising simultaneous or sequential oral and parenteral administration of said mammalian beta defensin.

  In one aspect, the invention relates to oral administration of mammalian beta-defensin for prophylactic treatment or as a method of keeping patients suffering from IBD in remission.

  In one aspect, the invention relates to prolonging this period of remission after oral administration of mammalian beta-defensin.

  In patients suffering from IBD, the original symptoms may occasionally return acutely, known as “relapse”. Depending on the situation, it may disappear spontaneously, but it may require medication. The time to relapse is between weeks and years and varies from patient to patient. In one embodiment of the invention, oral administration of mammalian beta-defensin reduces the incidence of relapse in patients suffering from IBD.

  As explained in the definitions section of this application, IBD is a group of inflammatory conditions of the colon and small intestine. Major types of IBD include Crohn's disease and ulcerative colitis (UC). In a preferred embodiment of the invention, the IBD to be treated is Crohn's disease and ulcerative colitis with mild activity and / or moderate to severe activity.

  In one embodiment of the present invention, oral administration of mammalian beta-defensin may be used to reduce neutrophil infiltration in the colonic epithelial lining. This decrease can be located in the proximal or distal colon.

  In other embodiments of the invention, oral administration of mammalian beta defensin can be used to treat inflammation in the colon.

  In yet another aspect of the invention, oral administration of mammalian beta-defensin can be used to regulate cytokine production in the epithelial lining in the gastrointestinal tract. The modulation is preferably a down-regulation of the activity of at least one cytokine selected from the group consisting of inflammatory cytokines and chemokines. Preferably, the modulation is down-regulating the activity of at least one cytokine selected from the group consisting of inflammatory IL-23, IL-1β, and TNFα, and / or up-regulating the activity of the anti-inflammatory cytokine IL-10 Control.

Transition of the disease activity index (DAI) score (by weight) under study. The results are shown as the average value of n = 10 animals per group ± standard deviation of the average value. Significant differences from the control (vehicle) group in specific data are shown as * P <0.05, ** P <0.01, *** P <0.001 (Kruskal-Wallis test for nonparametric data). Histological score of proximal colon sample. The results are shown as the average value of n = 10 animals per group ± standard deviation of the average value. Significant differences from the control (vehicle) group are shown as ** P <0.01, *** P <0.001 (Kruskal-Wallis test + Dunn's posttest for nonparametric data). Histological score of distal colon sample. The results are shown as the average value of n = 10 animals per group ± standard deviation of the average value. Significant differences from the control (vehicle) group are indicated as ** P <0.01 (Kruskal-Wallis test + Dunn's posttest for nonparametric data). Clustal W (2.1) multiple sequence alignment of beta defensin 2 of human, rhesus monkey, chimpanzee and orangutan. * Indicates a position with a single complete conserved residue. : Indicates that one of the following “strong” groups is fully conserved: -STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW. Indicates that one of the following “weak” groups is fully conserved: -CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY. Clustal W (2.1) multiple sequence alignment of human defensin 1 to 4. * Indicates a position with a single complete conserved residue. : Indicates that one of the following “strong” groups is fully conserved: -STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW. Indicates that one of the following “weak” groups is fully conserved: -CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.

  Definition

  Defensin: As used herein, the term “defensin” refers to a polypeptide that is recognized by those skilled in the art as belonging to the defensin class of antimicrobial peptides. To determine whether a polypeptide is a defensin according to the present invention, the amino acid sequence can be compared to the hidden Markov model profile (HMM profile) of the PFAM database by using the freely available HMMER software package.

  The PFAM defensin family includes, for example, Defensin_1 or “mammalian defensin” (accession number PF00323) and Defensin_2 or Defensin_beta or “beta defensin” (accession number PF00711).

  The defensin of the present invention belongs to the beta defensin class. Defensins from the beta defensin class share common structural features known to those skilled in the art, such as cysteine patterns.

  Examples of defensins according to the invention include human beta defensin 1 (hBD1, see SEQ ID NO: 1), human beta defensin 2 (hBD2, see SEQ ID NO: 2), human beta defensin 3 (hBD3, see SEQ ID NO: 3), human beta Defensin 4 (hBD4, see SEQ ID NO: 4) and mouse beta defensin 3 (mBD3, see SEQ ID NO: 6) are included.

  Identity: The relationship between two amino acid sequences or two nucleotide sequences is described by the parameter “identity”.

For the purposes of the present invention, the degree of identity between two amino acid sequences is preferably determined from the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277; http://emboss.org) using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) . The optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the needle, labeled “longest identity (obtained using the -nobrief option)” is used as percent identity and is calculated as follows:
(Same residue x 100) / (Length of alignment-Total number of gaps in alignment)

  Isolated polypeptide: As used herein, the term “isolated variant” or “isolated polypeptide” refers to a variant or polypeptide isolated from a source. In one embodiment, the variant or polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least pure by RP-HPLC analysis. 40%, more preferably at least 60% purity, even more preferably at least 80% purity, most preferably at least 90% purity.

  Substantially pure polypeptide: The term “substantially pure polypeptide” as used herein refers to other polypeptide materials that are naturally or recombinantly associated with at most 10% by weight, Preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably Refers to a polypeptide preparation containing at most 1%, more preferably at most 0.5%. Thus, a substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably, by weight relative to the total polypeptide material present in the preparation. At least 96% purity, more preferably at least 96% purity, more preferably at least 97% purity, more preferably at least 98% purity, even more preferably at least 99% purity, most preferably at least 99.5% purity, even more preferably At least 100% purity. The polypeptide of the present invention is preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods, classical purification methods, or chemical synthesis.

  Remission: As used herein, the term “remission” refers to the period of time during which the disease is controlled, for an active disease often referred to as “seizure”.

  Relapse: The term “relapse” as used herein refers to the reoccurrence of symptoms of IBD. The most common early symptoms of IBD are chronic diarrhea (sometimes hemorrhagic), convulsive abdominal pain, fever, loss of appetite, and weight loss. Symptoms last for days or weeks and may disappear without treatment. IBD recurs throughout the life of the patient at irregular intervals. Relapses may be mild or severe, short-term or long-term. Severe recurrence can lead to severe pain, dehydration, and blood loss.

  Inflammatory bowel disease (IBD): Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine. Major types of IBD include Crohn's disease and ulcerative colitis (UC).

Other forms of IBD are much less common and are not always classified as typical IBD:
・ Collagen colitis ・ Lymphocytic colitis ・ Ischemic colitis ・ Fecal flow-changing colitis ・ Behcet's disease ・ Indeterminate colitis

  The main difference between Crohn's disease and UC is the location and nature of inflammatory changes. Crohn's disease can affect any part of the digestive tract from the mouth to the anus (skin lesions), but the majority of cases begin at the terminal ileum. On the other hand, ulcerative colitis is limited to the colon and rectum.

  Treatment: As used herein, the terms “treatment” and “treat” refer to the management and care of a patient aimed at combating a condition, disease or disorder. Includes any treatment for a particular condition that afflicts the patient, such as alleviating or reducing symptoms or complications, delaying the progression of the condition, disease or disorder, and curing or eliminating the condition, disease or disorder And / or administration of an active compound for the purpose of preventing a condition, disease or disorder, wherein “preventing” or “preventing” prevents or reduces the occurrence of a condition, disease or disorder, Or should be understood to indicate patient management and care aimed at delaying, including administration of active compounds to prevent or reduce the risk of developing symptoms or complications. The patient to be treated is preferably a mammal, in particular a human. Patients to be treated according to the present invention can be of various ages.

  Mammalian beta-defensins

  The present invention relates to a mouse, monkey, or mammalian beta defensin such as human beta defensin, more preferably a human family defensin, more preferably human beta defensin in the treatment of inflammatory bowel diseases such as ulcerative colitis and / or Crohn's disease. Relating to the pharmaceutical use of

  In one embodiment, the mammalian beta defensin of the present invention is at least against any of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 6. It has a degree of identity of 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%. In a preferred embodiment, the mammalian beta defensin of the present invention is at least 80%, preferably at least 85, relative to any of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and / or SEQ ID NO: 4. %, More preferably at least 90%, most preferably at least 95%. In a further preferred embodiment, the mammalian beta defensin of the present invention comprises human beta defensin 1 (SEQ ID NO: 1), human beta defensin 2 (SEQ ID NO: 2), human beta defensin 3 (SEQ ID NO: 3), human beta defensin 4 ( SEQ ID NO: 4), human beta defensin 4 variant (SEQ ID NO: 5), and / or mouse beta defensin 3 (SEQ ID NO: 6). In an even more preferred embodiment, the mammalian beta defensin of the invention is human beta defensin 1 (SEQ ID NO: 1), human beta defensin 2 (SEQ ID NO: 2), human beta defensin 3 (SEQ ID NO: 3), and / or human beta It consists of defensin 4 (SEQ ID NO: 4).

  In a preferred embodiment, the mammalian beta defensin of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% identity to the amino acid sequence of SEQ ID NO: 2. Have a degree of In a preferred embodiment, the mammalian beta defensin of the present invention consists of human beta defensin 2 (SEQ ID NO: 2).

  In yet another embodiment, the mammalian beta defensin of the present invention consists of human beta defensin and / or mouse beta defensin and functionally equivalent variants thereof. Preferably, the mammalian beta defensin is functionally equivalent to human beta defensin 1, human beta defensin 2, human beta defensin 3, human beta defensin 4, chimpandi beta defensin 2, macaque beta defensin 2, and mouse beta defensin 3. It consists of various mutants. More preferably, the mammalian beta defensin of the present invention consists of human beta defensin 2 and a functionally equivalent variant thereof.

  The mammalian beta defensin of the present invention is also referred to as a preferred embodiment compound.

  In the present invention, a `` functionally equivalent variant '' of a mammalian (e.g., human) beta defensin is a modified mammal (e.g., a human that exhibits substantially the same effect as a parent mammal (e.g., human) beta defensin on inflammatory bowel disease). ) Beta defensin. Preferably, it also has substantially the same effect as mammalian (eg human) beta defensin on the activity of inflammatory cytokines and chemokines and / or anti-inflammatory cytokines. In one embodiment, the effect on the activity of inflammatory and anti-inflammatory cytokines is measured as described in Examples 4 and 6 in WO2010 / 007166. The effect is a human cell selected from the group consisting of human peripheral blood mononuclear cells (PBMC), human CD14 + monocyte-derived dendritic cells, human monocyte cell lines, and human immature dendritic cells, preferably human PBMC Can be measured.

  According to the present invention, a functionally equivalent variant of a mammalian (eg human) beta defensin preferably has a human beta defensin 2 having SEQ ID NO: 2 compared to the amino acid sequence of a mammalian (eg human) beta defensin. In comparison, 1 to 5 amino acid modifications, preferably 1 to 4 amino acid modifications, more preferably 1 to 3 amino acid modifications, most preferably 1 to 2 amino acid modifications, in particular 1 amino acid modification Can be included.

  The term “modification” as used herein means any chemical modification of a mammalian (eg, human) beta defensin. Modifications can be amino acid substitutions, deletions and / or insertions, amino acid side chain exchanges, or the use of unnatural amino acids with similar properties in the amino acid sequence. In particular, the modification can be amidation, such as C-terminal amidation.

  Preferably, the amino acid modification is of a minor nature, i.e., conservative amino acid substitutions or insertions, single deletions, minor amino acids or carboxyls without significant effect on polypeptide folding and / or activity. A small extension that facilitates purification by altering the net charge, such as a terminal extension or polyhistidine tag, antigenic epitope, or binding domain. In one embodiment, a small extension, such as a polyhistidine tag, antigenic epitope, or binding domain is attached to a mammalian (e.g., human) beta defensin via a small linker peptide of up to about 20-25 residues, The linker may include a restriction enzyme cleavage site. The Clustal W alignment of FIG. 4 or FIG. 5 can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein. The sequences were aligned using Clustal W 2.1 (http://www.genome.jp/tools/clustalw/) and the following settings: gap opening penalty: 10, gap extension penalty: 0,05, weight transition: NO , Hydrophilic residue for protein: GPSNDQE, hydrophilic gap: YES, weight matrix: BLOSUM (for PROTEIN).

Substitutions within the following groups (Clustal W, “strong” conservation group) should be considered as conservative substitutions within the meaning of the present invention.
-STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Substitution within the following groups (Clustal W, “weak” conserved group) should be considered a semi-conservative substitution in the sense of the present invention.
-CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.

  Examples of conservative substitutions are basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic Substitutions made within the group of family amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, in H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

  In addition to the 20 standard amino acids, non-standard amino acids (4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) are replaced with amino acid residues of the wild-type polypeptide Can do. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids can be substituted for amino acid residues. “Non-natural amino acids” are modified after protein synthesis and / or have a structure in the side chain (s) that differs from standard amino acids. Non-natural amino acids are chemically synthesizable and are preferably commercially available and include pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

  Essential amino acids in mammalian beta-defensins can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter approach, a single alanine mutation is introduced into every residue in the molecule and the resulting mutant molecule is biologically active (i.e., activity against inflammatory bowel disease and / or TNF-α). Activity inhibition) to identify amino acid residues that are important for the activity of the molecule. See Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The identity of the essential amino acids can also be inferred from analysis of identity with the polypeptide for mammalian beta defensin (see Clustal W alignments in FIGS. 4 and 5).

  Single or multiple amino acid substitutions can be performed using known mutagenesis, recombination, and / or shuffle methods followed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57, Bowie and Sauer, 1989. Natl. Acad. Sci. USA 86: 2152-2156, WO95 / 17413, or WO95 / 22625, and can be made and tested by related screening procedures. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837, US Patent No. 5,223,409, WO 92/06204), and region-specific mutagenesis. (Derbyshire et al., 1986, Gene 46: 145, Ner et al., 1988, DNA 7: 127).

  The N-terminal extension of the polypeptide of the present invention may suitably be composed of 1 to 50 amino acids, preferably 2 to 20 amino acids, especially 3 to 15 amino acids. In one embodiment, the N-terminal peptide extension does not include Arg (R). In other embodiments, the N-terminal extension comprises a kex2 or kex2-like cleavage site as defined below. In a preferred embodiment, the N-terminal extension is a peptide comprising at least two Glu (E) and / or Asp (D) amino acid residues, for example, an N-terminal extension comprising one of the following sequences: : EAE, EE, DE, and DD.

  If the outcome of a particular substitution cannot be reliably predicted, the derivative can be readily assayed by the methods described above to determine the presence or absence of biological activity.

  Methods and uses

  Human beta-defensin 2 significantly reduces the severity of disease parameters in a 10-day dextran sulfate sodium (DSS) -induced colitis model in mice, thus treating inflammatory bowel diseases such as ulcerative colitis and Crohn's disease It has been found to exhibit potent activity as a drug for.

  Thus, the present invention provides a method for the prophylactic treatment of inflammatory bowel disease or a method for keeping patients suffering from IBD in remission, which can be used to treat human beta defensin 2 and the like for patients in need of such treatment. Orally administering an effective amount of a mammalian beta-defensin, eg, in the form of a pharmaceutical composition.

  In one embodiment, the invention relates to extending the duration of this remission after oral administration of mammalian beta-defensin or reducing the occurrence of relapses in patients suffering from IBD.

  The inflammatory bowel disease according to the invention relates to Crohn's disease and ulcerative colitis. In a preferred embodiment, the stage of Crohn's disease to be treated with oral administration of mammalian beta-defensin is Crohn's disease with remission and / or mild activity. In other preferred embodiments, the stage of ulcerative colitis to be treated with oral administration of mammalian beta-defensin is the period of remission of ulcerative colitis (e.g., total colitis / remission, left colitis / Remission and refractory disease in remission).

  In one embodiment of the invention, oral administration of mammalian beta-defensin may be used to reduce neutrophil infiltration in the colonic epithelial lining. This decrease can be located in the proximal or distal colon.

  In other embodiments of the invention, oral administration of mammalian beta-defensin may be used for the treatment of inflammation in the colon.

  In still other embodiments of the invention, oral administration of mammalian beta-defensin may be used to modulate cytokine production in the epithelial lining in the gastrointestinal tract.

  The modulation is preferably a down-regulation of the activity of at least one cytokine selected from the group consisting of inflammatory cytokines and chemokines. Preferably, the modulation is down-regulating the activity of at least one cytokine selected from the group consisting of inflammatory IL-23, IL-1β, and TNFα, and / or up-regulating the activity of the anti-inflammatory cytokine IL-10 Control.

  In other embodiments, the present invention provides a method of treating inflammatory bowel disease, the treatment comprising simultaneous or sequential parenteral and oral administration of an effective amount of mammalian beta-defensin in the form of a pharmaceutical composition. Including doing. During an active outbreak of inflammatory bowel disease (`` seizure ''), a patient suffering from IBD, in one embodiment, is given parenteral administration of mammalian beta-defensins, such as human beta-defensin 2, in combination with or subsequent to this. Then, oral administration of the mammalian beta-defensin during remission.

  In one embodiment, a method of treatment with hBD2 results in significant suppression of disease activity index (DAI) in an in vivo model of IBD when administered by oral route twice daily at three different dose levels, where hBD2 is Furthermore, it showed a very significant anti-inflammatory activity in the model.

  Mammalian beta defensin can be utilized therapeutically in compositions formulated for administration by any conventional route. In one embodiment of the invention, mammalian beta defensin is administered orally.

  In other embodiments of the invention, the mammalian beta defensin is administered by simultaneous or sequential oral and parenteral administration.

  Oral administration is usually done for drug delivery in the intestine and the agent is delivered through the intestinal mucosa.

  Parenteral administration is any route of administration that is not an oral / intestinal route, which avoids first-pass degradation of the drug in the liver. Thus, parenteral administration includes any injection and infusion, for example, bolus injection or continuous infusion, such as intravenous administration, intramuscular administration, subcutaneous administration.

  Subcutaneous and intramuscular forms of parenteral administration are generally preferred.

  In still other embodiments, the compositions of preferred embodiments may be formulated as lyophilizates utilizing appropriate excipients that provide stability as lyophilizate and stability after rehydration. .

  Pharmaceutical compositions containing mammalian beta defensin, such as human beta defensin, can be manufactured by conventional methods such as mixing, granulating, coating, dissolving, or lyophilizing processes.

  A preferred embodiment pharmaceutical composition comprises a mammalian beta defensin, such as human beta defensin, and a pharmaceutically acceptable carrier and / or diluent.

  Pharmaceutically acceptable carriers and / or diluents are well known to those skilled in the art. For compositions formulated as solutions, acceptable carriers and / or diluents include saline, sterile water, and optional antioxidants, buffers, bacteriostats, and other common additives. Can be included.

  Parenteral administration may be in the form of injections and infusions, and the formulation may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the active ingredient may be in the form of a powder obtained by aseptic isolation of a sterile solid or lyophilization from a solution as a composition prior to use with a suitable vehicle, eg, sterile pyrogen-free water. The formulation can be provided in unit-dose or multi-dose enclosed containers such as ampoules, vials, prefilled syringes, infusion bags, or the addition of sterile liquid excipients for injection, such as water, just prior to use. Can be stored in a freeze-dried state that requires only

  Examples of oily or non-aqueous carriers, diluents, solvents, or vehicles include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, preservatives, wetting agents, emulsifying agents, or suspending agents. , Stabilizers and / or dispersants and the like.

  The compounds of the present invention may be formulated in various dosage forms for oral administration. Solid preparations can include powders, tablets, drops, capsules, troches, and dispersible granules. Other forms suitable for oral administration are emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpastes, gel dentifrices, liquid preparations including chewing gum, or solutions, suspensions shortly before use. And solid preparations for conversion to liquid preparations such as emulsions.

  In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active ingredient is mixed with the carrier having the necessary binding capacity in suitable proportions and compressed into the desired shape and size. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.

  The drop according to the present invention comprises a sterile or non-sterile aqueous or oily solution or suspension, dissolves the active ingredient in a suitable aqueous solution, optionally a disinfectant and / or antifungal agent and / or any other suitable It can be prepared by including a surfactant, optionally including a preservative. Suitable solvents for the preparation of an oily solution include glycerin, diluted alcohol, and propylene glycol.

  The emulsion may be prepared in solution in an aqueous propylene glycol solution or may contain an emulsifier such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with natural or synthetic rubber, resin, viscous materials such as methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents.

  Formulations (in addition to mammalian beta-defensins and any other active ingredients) carriers, extenders, disintegrants, lubricants, sugars and sweeteners, flavors, preservatives, stabilizers, wetting agents, emulsifiers, solubilization Agents, salts for adjusting osmotic pressure, buffers, diluents, dispersants and surfactants, binders, lubricants, and / or other pharmaceutical additives known in the art.

  One skilled in the art can further formulate mammalian beta-defensins in appropriate forms according to common practice, such as those described in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, PA 1990. .

  Mammalian beta defensin, such as human beta defensin, alone or in combination therapy with one or more other pharmaceutical compounds or drug substances and / or one or more pharmaceutically acceptable excipients Can be used with the agent.

  In vitro synthesis

  Mammalian beta defensin can be prepared by in vitro synthesis using conventional methods known in the art. Various commercially available synthesizers can be used. For example, automatic synthesizers such as Applied Biosystems Inc. and Beckman can be used. By using a synthesizer, naturally occurring amino acids are unnatural amino acids, especially D-form (or D-form), such as D-alanine and D-isoleucine, diastereoisomers, side chains with different lengths or functionality, etc. Can be substituted. The specific sequence and method of preparation are determined by convenience, economy, required purity, and the like.

  Various chemical linkages with convenient functions for conjugation, such as amino groups for amide or substituted amine formation, such as amino groups for reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, etc. Can be provided for any peptide or protein.

  If desired, various groups can be introduced into the peptide that allow linkage to other molecules or surfaces during synthesis or expression. Accordingly, cysteine can be used to create a thioether, a histidine for linking with a metal ion complex, a carboxyl group for forming an amide or ester, an amino group for forming an amide, and the like.

  Mammalian beta defensin or its functional equivalent may be isolated and purified by conventional recombinant synthetic methods. Recombinant synthesis can be performed using appropriate expression vectors and eukaryotic expression systems. Solutions can be prepared from expression hosts and media, and the existing defensins can be purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. A method for recombinant expression of human beta-defensin 2 in E. coli is disclosed in WO2010 / 007166 (Novozymes).

  Dose

  Mammalian beta defensin, such as human beta defensin, is preferably utilized in a pharmaceutical composition in an amount effective to treat inflammatory bowel disease, preferably in a state that is toxic to the patient. For such treatment, the appropriate dosage will, of course, be dependent on, for example, the chemical nature and pharmacokinetic data of the compounds of the invention used, the particular host, the mode of administration and the nature and severity of the condition being treated. Will change accordingly. In general, however, the indicated daily dose is preferably from about 0.0001 mg / kg body weight to about 10 mg / kg body weight, more preferably about 0.001 mg / kg body weight in order to obtain satisfactory results in many mammals, such as humans. kg body weight to about 10 mg / kg body weight, for example 0.005 mg / kg body weight to 5 mg / kg body weight, more preferably about 0.01 mg / kg body weight to about 10 mg / kg body weight, preferably about 0.1 mg / kg body weight to about 10 mg / kg body weight, eg, administered in divided doses up to once, twice, three times, or four times a day. The compounds of the preferred embodiments can be administered to large mammals such as humans in the same dosage regimen at the same dosages as conventionally used.

  In one embodiment, the daily dose is preferably 0.0001 to 10 mg / kg body weight, more preferably 0.001 to 10 mg / kg body weight, more preferably 0.005 to 5 mg / kg body weight.

  In certain embodiments, a pharmaceutical composition of a preferred embodiment comprises about 0.5 mg or less to about 1500 mg or more, preferably about 0.5 mg / unit dosage form of mammalian beta defensin, such as human beta defensin, depending on the route of administration. 0.6, 0.7, 0.8, or 0.9 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg, more preferably about 1, 2, 3, 4 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg to about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or Can be included in an amount of 100 mg. However, in certain embodiments, lower or higher dosages than those described above may be preferred. Appropriate concentrations and dosages can be readily determined by one skilled in the art.

  In one embodiment, the mammalian beta defensin is administered at least once a day, such as at least twice a day, such as at least three times a day, such as at least four times a day.

  The invention is further described by the following examples which should not be construed as limiting the scope of the invention.

  Example 1

  A 10-day dextran sulfate sodium (DSS) -induced colitis model in mice

  The purpose of the following study was to determine the anti-inflammatory activity of human beta-defensin 2 in an acute (10-day) model of inflammatory bowel disease (colitis) induced by oral dextran sulfate sodium (DSS) administration in mice. there were.

  The DSS colitis mouse model is a well-known model for the study of inflammatory bowel disease. Kawasada et al. "Insights from advances in research of chemically induced experimental models of human inflammatory bowel disease", World J. Gastroenterol. , Vol. 13 (42), pp. 5581-5593 (2007), and Wirtz and Neurath "Mouse models of inflammatory bowel disease", Advanced Drug Delivery Reviews, Vol. 59 (11), 1073-1083 (2007). Has been.

  material

  Test item

  Human beta defensin 2 (hBD2), methylprednisolone 21-hemisuccinate ("prednisolone"), PBS buffer (GIBCO).

  Experimental animals

  Male C57BL / 6 mice (Harlan Interfauna Iberica, Barcelona, Spain) are of a species and sex that has been shown to cause significant inflammation of the colon when administered with a 2% solution of DSS in drinking water for 10 days. Because of this, it was used for research.

  specific

  Animals were identified by tail number and letter code. In addition, each cage was identified by a color-coded card indicating the animal number and gender, test article code or name, dose level, route of administration, treatment period, group number, study code, and study supervisor name.

  weight

  The average body weight of the animals on the study start date was 22.4 ± 0.16 g.

  Acclimatization

  Before the start of the study, the conditions were the same as those for the main study for a minimum of 7 days.

  Containment

  Upon arrival, animals were housed randomly in polycarbonate cages (E type, Charles River, 255 × 405 × 197 mm) with stainless steel lids. Animals should be sexed in an animal room with controlled temperature (22 ± 2 ° C), lighting (light / dark 12/12 hours), air pressure, number of air changes, and relative humidity (30-70%) Housed in groups of 5 per cage. All cages were covered with sawdust (Lignocel 3-4, Harlan Interfauna Iberica, Spain) on the floor. All mice had free access to a standard dry rodent diet (Teklad Global 2014, Harlan Interfauna Iberica, Spain). Water was provided in bottles as appropriate. The supply of tap water to the animal room was analyzed periodically, the composition was confirmed, and possible contaminants (chemicals and microorganisms) were detected.

  Equipment and materials

Instrument:
Animal scale Sartorius model BP 2100
Surgical cutting instrument
Eppendorf 5415C centrifuge
Nikon Eclipse E600FN microscope
Hook & Tucker instruments rotamixer
IKA Ultra Turrax homogenizer
Sartorius model BP 221S chemical balance

Materials and reagents:
Sterile disposable syringe (1 ml)
Sterile Butterfly 25G Injection Set Anesthetic (Ketamine / Xylazine)
Local anesthetic cream (EMLA, Astra Zeneca)
Dextran sulfate sodium 30,000-50,000 Da (MP Biomedicals)
Phosphate buffered saline (PBS, Sigma)
Neutral buffered formalin (VWR)

  Experimental protocol

  Study design: Animals were divided into 5 experimental groups. Each group consisted of 10 males. Group A: treated with oral administration twice daily with control vehicle (PBS). Group B: treated with methylprednisolone (1 mg / kg orally administered twice a day). Group C: treated with hBD2 (0.05 mg / kg orally administered twice a day). Group D: treated with hBD2 (0.5 mg / kg orally administered daily). Group E: treated with hBD2 (daily oral administration of 5 mg / kg). The assignment of animals to all experimental groups was random. A maximum of 5 animals were housed in each cage (as per Directive86 / 609 / EEC). All animals were weighed in the laboratory upon arrival before administration of the test article.

  Administration of test substance

  The control vehicle and hBD2 were orally administered at a dose of 10 ml / kg body weight using a sterile cannula. All groups of animals received the corresponding test article (test, reference, or vehicle) twice a day (9.00 hours and 17.00 hours) for 10 consecutive days (experimental days 1-10).

  Experimental procedure

  Induction of colitis

  Colitis was induced in mice by adding 2% DSS to drinking water for 7 days. On day 1 all mice were weighed and marked according to experimental group. DSS solution was put into the beverage bottles of each cage, and the lids of all the bottles were correctly attached to confirm that there was no clogging. On the third day, the solution remaining in the bottle was discarded and a new DSS solution was added. This procedure was repeated on day 5. On day 8, the remaining solution was discarded and replaced with autoclaved water. The animals were sacrificed on day 10 after 2 days.

  Clinical evaluation (disease activity index)

  Daily clinical evaluation of DSS-treated animals was performed by calculating an effective clinical disease activity index (DAI) ranging from 0 to 4 with the following parameters: stool consistency, presence of rectal bleeding, And weight loss.

  Weight loss was calculated as the percent difference between the original weight (Day 1) and the actual weight on each experimental day (Day 2-10). The occurrence of diarrhea was defined as mucus / fecal matter attached to the anal hair. Rectal bleeding was defined as diarrhea with visible blood / mucus or gross rectal bleeding. The maximum daily score for DAI is 12.

  Euthanasia and collection of colon samples

  On day 10, two hours after the last dose of control vehicle, hBD2, or prednisolone, the animals were killed by overdose of anesthetic. The colon was removed and the length and weight were measured after removal of the cecum. Two parts of the colon (proximal and distal) were removed from each animal, stored in neutral buffered formalin, and then histologically analyzed by the following scoring method (hematoxylin and eosin staining):

  Descriptive score: No change is observed (0). There is microscopic scattered mucosal inflammatory cell infiltration, with or without microscopic epithelial hyperplasia (1). In some cases, it extends into the submucosa and has mild to diffuse inflammatory cell infiltration with wrinkles, with micro to mild epithelial hyperplasia and micro to mild goblet cell mucin deposition (2 ). It is transmural in some cases and has mild to moderate inflammatory cell infiltration, often accompanied by ulcers, with moderate epithelial hyperplasia and mucin deposition (3). It is often transmural, has strong inflammatory cell infiltration with ulcers, and has strong epithelial hyperplasia and mucin deposition (4). There is intense inflammatory cell infiltration with severe ulcers and intestinal gland loss (5).

  Histological evaluation

  Two parts of the colon (proximal and distal) were removed from each animal, processed for histological analysis (hematoxylin and eosin staining) and scored by a blinded observer according to the histological scoring method described above.

  Statistical analysis

  The statistical significance of the results was evaluated using the statistical program GraphPad Instat3. Results are shown as mean ± standard deviation of mean, where (n) is the number of animals. Differences between groups in body weight, colon length, and colon weight were assessed by Dunnett's post-test in addition to analysis of variance (ANOVA) on unmatched data, allowing multiple comparisons. Differences between groups in disease activity index and histological score were evaluated by multiple comparisons by Kruskal-Wallis test + Dunnet's post-test on unmatched data. A value of P <0.05 was considered significant. In each figure and table, significant differences from the corresponding control (vehicle) groups are shown as * P <0.05, ** P <0.01, *** P <0.001.

  result

  In this study, treatment with the test article NZ39000 (hBD2) was performed daily at three different dose levels (0.05, 0.5, and 5 mg / kg) when administered by the oral route in the Disease Activity Index (DAI). It resulted in significant suppression (Figure 1 and the table below). Significant suppression was observed both with the following significant differences after treatment with the reference product (prednisolone) and hBD2.

  There were no significant differences between the unlisted days and treatments.

  Significant reduction was observed in both the proximal and distal colon (Figures 2 and 3).

  Conclusion

  The results show that hBD2 administered orally at the test dose increased the increase in disease activity index induced by DSS administration at 0.05 mg / kg on days 6, 7, 8, 9, and 10 and at 0.5 mg / kg. On days 6, 9, and 10, it has been demonstrated that 5 mg / kg significantly decreases on days 3, 4, 5, 6, 7, 8, 9, and 10. Similar to the results obtained with the disease activity index, histological analysis of the proximal and distal colons of each animal on day 10 revealed a very significant decrease in tissue damage score with orally administered hBD2. became. The effect was more prominent at the proximal than at the distal. In this study, treatment with test article hBD2 resulted in a significant suppression of disease activity index (DAI) when administered by oral route daily at three dose levels. The reference product (prednisolone) is known to have a significant appetite-suppressing effect in mice (administered twice daily) and may therefore have a significant effect on animal weight regardless of disease severity There is. In view of this effect, additional DAI scores were performed with no change in body weight effect, and only stool stiffness and rectal bleeding were used as measurable parameters. In this case, the test product hBD2 showed a very significant anti-inflammatory activity similar to the reference product prednisolone. Administration of dextran sulfate sodium resulted in significant inflammation and damage of the animal's colon tissue, as evidenced after histological examination. This damage was more pronounced in the distal colon when compared to the proximal part. Treatment with the test article hBD2 twice daily at three dose levels resulted in a significant reduction in tissue damage in the proximal colon. Similarly, treatment at medium and high dose levels twice daily with test article hBD2 further resulted in a significant reduction in tissue damage in the distal colon. Treatment with prednisolone at a dose of 1 mg / kg orally twice daily (Group B) also significantly reduced tissue damage induced by DSS in the proximal and distal colon. The results further show a significant weight gain at day 10 in animals treated with the high dose test article when compared to the control group (P <0.05). This result suggests an improvement in the overall health status of the animals (eg, due to increased food intake, decreased diarrhea, etc.) in the test product group described above, resulting in decreased weight loss. Animals treated with prednisolone (Group B) show a significant decrease in body weight from day 2 to the end of the study. As mentioned above, this weight loss cannot be considered as an indication of the overall health of the animal in this particular case, and prednisolone may have a significant inhibitory effect on the appetite of mice. Known and this accounts for weight loss. The results obtained in this study demonstrate the anti-inflammatory activity of orally administered hBD2 in a model of DSS colitis induced in mice after a 10-day treatment period.

Claims (101)

  Use of mammalian beta-defensins in the manufacture of medicaments for the treatment of inflammatory bowel disease that are administered orally and parenterally simultaneously or sequentially.   The use of claim 1, wherein the drug is administered orally after parenteral administration of the drug at least once.   The use according to claim 1, wherein the medicament is administered orally together with at least one parenteral administration of the dose of the medicament.   4. Use according to claims 1 to 3, wherein certain defensins are administered orally, certain defensins are administered parenterally and the orally administered defensins and parenterally administered defensins are the same.   The use according to claims 1 to 3, wherein a given defensin is administered orally, and a given defensin is administered parenterally, wherein said orally administered defensin and parenterally administered defensin are different.   Use of mammalian beta-defensins in the manufacture of orally administered drugs for the prophylactic treatment of inflammatory bowel disease.   Use of mammalian beta-defensins in the manufacture of orally administered drugs to keep inflammatory bowel disease in remission.   Use of mammalian beta-defensins in the manufacture of orally administered drugs to prolong remission of inflammatory bowel disease.   Use of mammalian beta-defensins in the manufacture of orally administered drugs to reduce the occurrence of recurrence of inflammatory bowel disease.   The use according to any one of the preceding claims 6 to 9, wherein the inflammatory bowel disease is Crohn's disease.   11. Use according to claim 10, wherein the Crohn's disease is a mild active disease or a moderate to severe active disease.   10. The use according to any one of claims 6 to 9, wherein the inflammatory bowel disease is ulcerative colitis.   13. The method according to claim 12, wherein the colitis is total colitis or left colitis.   Use of mammalian beta-defensins in the manufacture of orally administered drugs to reduce neutrophil infiltration in the colonic epithelial lining.   15. Use according to claim 14, wherein neutrophil infiltration is reduced in the proximal colon.   16. Use according to claim 15, wherein neutrophil infiltration further decreases in the descending and sigmoid colon.   Use of mammalian beta-defensins in the manufacture of orally administered drugs to reduce inflammation in the colon.   18. Use according to claim 17, wherein the inflammation in the colon is an inflammatory bowel disease.   Use of mammalian beta-defensins in the manufacture of orally administered drugs to regulate cytokine production in the epithelial lining of the gastrointestinal tract.   20. Use according to claim 19, wherein the cytokine production in the epithelial lining of the gastrointestinal tract results from inflammatory bowel disease.   The agent is effective in down-regulating the activity of at least one cytokine or chemokine selected from the group consisting of IL-23, IL-1β, IL-6, IL-8, MCP-1, and TNFα. Item 19. Use according to Item 19.   20. Use according to claim 19, wherein the medicament is effective in upregulating the activity of IL-10.   Use according to any preceding claim, wherein the medicament is administered to a human and the mammalian beta defensin is human beta defensin.   The preceding claim, wherein the mammalian beta defensin has at least 80% sequence identity to any of the amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Use as described in any of the above.   Use according to any preceding claim, wherein the mammalian beta defensin has at least 80% sequence identity to SEQ ID NO: 2.   The mammalian beta defensin is a variant of human beta defensin 2, wherein no more than 10, preferably no more than 5, more preferably no more than 2 amino acids have changed compared to SEQ ID NO: 2. Use according to any of the paragraphs.   The mammalian beta defensin is at least 1 in human cells selected from the group consisting of human PBMC, human CD14 + monocyte-derived dendritic cells, human monocyte cell lines, and human immature dendritic cells, preferably human PBMC. Use according to any preceding claim, wherein the activity of one inflammatory cytokine can be down-regulated.   28. Use according to claim 27, wherein the inflammatory cytokine is selected from the group consisting of TNFα, IL-23, and IL-1β, preferably TNFα.   The mammalian beta defensin is at least 1 in human cells selected from the group consisting of human PBMC, human CD14 + monocyte-derived dendritic cells, human monocyte cell lines, and human immature dendritic cells, preferably human PBMC. Use according to any of the preceding claims, wherein the activity of one anti-inflammatory cytokine can be upregulated.   30. Use according to claim 29, wherein the anti-inflammatory cytokine is IL-10.   Use according to any of the preceding claims, wherein the mammalian beta defensin is administered at least once a day, for example at least twice a day, for example at least three times a day.   The mammalian beta defensin is administered at a daily dose of 0.0001 to 10 mg / kg, preferably 0.001 to 10 mg / kg, more preferably 0.005 to 5 mg / kg. use.   Mammalian beta defensin used in the treatment of inflammatory bowel disease, wherein the method comprises simultaneous or sequential oral and parenteral administration of an effective amount of mammalian beta defensin.   34. The use of claim 33, wherein the defensin is administered orally after at least one parenteral administration of the defensin.   34. Use according to claim 33, wherein the defensin is administered orally together with at least one parenteral administration of a defensin dose.   34. Use according to claim 33, wherein a defensin is administered orally and a defensin is administered parenterally, the orally administered defensin and the parenterally administered defensin.   34. The use of claim 33, wherein a defensin is administered orally and a defensin is administered parenterally, wherein the orally administered defensin and the parenterally administered defensin are different.   A mammalian beta defensin used in the prophylactic treatment of inflammatory bowel disease, wherein the method comprises oral administration of an effective amount of mammalian beta defensin to a patient.   Mammalian beta defensin for use in keeping a patient suffering from inflammatory bowel disease in remission, wherein the method comprises oral administration of a therapeutically effective amount of mammalian beta defensin to the patient.   Mammalian beta defensin for use in prolonging remission in a patient suffering from inflammatory bowel disease, wherein the method comprises oral administration of a therapeutically effective amount of mammalian beta defensin to the patient.   Mammalian beta defensin for use in reducing the occurrence of recurrence in a patient suffering from inflammatory bowel disease, said method comprising oral administration of a therapeutically effective amount of mammalian beta defensin to said patient .   42. Use according to any of the preceding claims 38 to 41, wherein the patient suffers from Crohn's disease.   39. Use according to claim 38, wherein the patient suffers from a mild active disease or a moderate to severe active disease.   42. Use according to any of claims 38 to 41, wherein the patient suffers from ulcerative colitis.   45. The use according to claim 44, wherein the colitis is total colitis or left colitis.   A mammalian beta-defensin for use in reducing neutrophil infiltration in the colonic epithelial lining, said method comprising orally administering an effective amount of mammalian beta-defensin to a patient in need thereof Beta defensin.   49. Use according to claim 46, wherein neutrophil infiltration is reduced in the proximal colon.   48. Use according to claim 47, wherein neutrophil infiltration further decreases in the descending and sigmoid colon.   Mammalian beta defensin used in reducing inflammation in the colon, wherein the method comprises oral administration of a therapeutically effective amount of mammalian beta defensin to a patient.   50. Use according to claim 49, wherein the patient suffers from inflammatory bowel disease.   Mammalian beta defensin used in regulating cytokine production in the epithelial lining of the gastrointestinal tract, wherein the method comprises oral administration of an effective amount of mammalian beta defensin to a patient in need thereof.   52. Use according to claim 51, wherein said patient suffers from inflammatory bowel disease.   The amount is effective for downregulating the activity of at least one cytokine or chemokine selected from the group consisting of IL-23, IL-1β, IL-6, IL-8, MCP-1, and TNFα, Item 51. Use according to Item 51.   52. Use according to claim 51, wherein said amount is effective in upregulating the activity of IL-10.   55. Use according to any of claims 33 to 54, wherein the patient is a human and the mammalian beta defensin is human beta defensin.   The mammalian beta defensin has at least 80% sequence identity to any of the amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Use according to any of 54 to 54.   55. Use according to any of claims 33 to 54, wherein the mammalian beta defensin has at least 80% sequence identity to SEQ ID NO: 2.   The mammalian beta defensin is a variant of human beta defensin 2, wherein no more than 10, preferably no more than 5, more preferably no more than 2 amino acids have changed compared to SEQ ID NO: 2. Use according to any of 33 to 57.   The mammalian beta defensin is at least 1 in human cells selected from the group consisting of human PBMC, human CD14 + monocyte-derived dendritic cells, human monocyte cell lines, and human immature dendritic cells, preferably human PBMC. 59. Use according to any of claims 33 to 58, capable of down-regulating the activity of two inflammatory cytokines.   60. Use according to claim 59, wherein the inflammatory cytokine is selected from the group consisting of TNFα, IL-23, and IL-1β, preferably TNFα.   The mammalian beta defensin is at least 1 in human cells selected from the group consisting of human PBMC, human CD14 + monocyte-derived dendritic cells, human monocyte cell lines, and human immature dendritic cells, preferably human PBMC. 61. Use according to any of claims 33 to 60, capable of upregulating the activity of two anti-inflammatory cytokines.   62. Use according to claim 61, wherein the anti-inflammatory cytokine is IL-10.   63. Use according to any of claims 33 to 62, wherein the mammalian beta defensin is administered at least once a day, for example at least twice a day, for example at least three times a day.   64. The mammalian beta defensin is administered at a daily dose of 0.0001 to 10 mg / kg, preferably 0.001 to 10 mg / kg, more preferably 0.005 to 5 mg / kg. Use of description.   A method of treatment of inflammatory bowel disease comprising simultaneous or sequential oral and parenteral administration of an effective amount of a mammalian beta-defensin.   66. The method of claim 65, wherein the defensin is administered orally after at least one parenteral administration of the defensin.   66. The method of claim 65, wherein the defensin is administered orally with at least one parenteral administration of a defensin dose.   A method of prophylactic treatment of inflammatory bowel disease comprising orally administering an effective amount of mammalian beta-defensin to a patient.   A method of keeping a patient suffering from inflammatory bowel disease in remission, comprising oral administration of a therapeutically effective amount of mammalian beta-defensin to said patient.   A method of prolonging remission in a patient suffering from inflammatory bowel disease, comprising oral administration of a therapeutically effective amount of mammalian beta-defensin to said patient.   A method of reducing the occurrence of recurrence in a patient suffering from inflammatory bowel disease, comprising oral administration of a therapeutically effective amount of mammalian beta-defensin to said patient.   72. A method according to any of claims 68 to 71, wherein the patient suffers from Crohn's disease.   73. The method of claim 72, wherein the patient suffers from a mild active disease or a moderate to severe active disease.   72. The method of any of claims 68-71, wherein the patient suffers from ulcerative colitis.   75. The method of claim 74, wherein the colitis is total colitis or left-side colitis.   A method of reducing neutrophil infiltration in the colonic epithelial lining comprising orally administering an effective amount of mammalian beta-defensin to a patient in need thereof.   77. The method of claim 76, wherein neutrophil infiltration is reduced in the proximal colon.   78. The method of claim 77, wherein neutrophil infiltration further decreases in the descending and sigmoid colon.   A method of reducing inflammation in the colon, comprising oral administration of a therapeutically effective amount of mammalian beta-defensin to a patient.   80. The method of claim 79, wherein the patient suffers from inflammatory bowel disease.   A method of modulating cytokine production in the epithelial lining of the gastrointestinal tract, comprising orally administering an effective amount of mammalian beta-defensin to a patient in need thereof.   84. The method of claim 81, wherein the enteral administration is oral.   82. The method of claim 81, wherein the patient suffers from inflammatory bowel disease.   The amount is effective for downregulating the activity of at least one cytokine or chemokine selected from the group consisting of IL-23, IL-1β, IL-6, IL-8, MCP-1, and TNFα, Item 81. The method according to Item 81.   92. The method of claim 81, wherein the amount is effective for upregulation of IL-10 activity.   86. The method of any of claims 68 to 85, wherein the patient is a human and the mammalian beta defensin is human beta defensin.   The preceding claim, wherein the mammalian beta defensin has at least 80% sequence identity to any of the amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The method according to any one of 68 to 86.   88. The method of any of claims 68-87, wherein the mammalian beta defensin has at least 80% sequence identity to SEQ ID NO: 2.   The mammalian beta defensin is a variant of human beta defensin 2, wherein no more than 10, preferably no more than 5, more preferably no more than 2 amino acids have changed compared to SEQ ID NO: 2. Item 89. The method according to any one of Items 68 to 88.   The mammalian beta defensin is at least 1 in human cells selected from the group consisting of human PBMC, human CD14 + monocyte-derived dendritic cells, human monocyte cell lines, and human immature dendritic cells, preferably human PBMC. 90. A method according to any of claims 68 to 89, wherein the activity of one inflammatory cytokine can be downregulated.   92. The method of claim 90, wherein the inflammatory cytokine is selected from the group consisting of TNFα, IL-23, and IL-1β, preferably TNFα.   The mammalian beta defensin is at least 1 in human cells selected from the group consisting of human PBMC, human CD14 + monocyte-derived dendritic cells, human monocyte cell lines, and human immature dendritic cells, preferably human PBMC. 92. The method according to any of claims 68 to 91, wherein the activity of one anti-inflammatory cytokine can be upregulated.   94. The method of claim 92, wherein the anti-inflammatory cytokine is IL-10.   94. A method according to any of the preceding claims 68 to 93, wherein the mammalian beta defensin is administered at least once a day, for example at least twice a day, for example at least three times a day.   95. Any of the preceding claims 68 to 94, wherein said mammalian beta defensin is administered at a daily dose of 0.0001 to 10 mg / kg, preferably 0.001 to 10 mg / kg, more preferably 0.005 to 5 mg / kg. The method described in 1.   96. A mammalian beta defensin for use in any of the methods of the preceding claims 68-95.   A method of administering mammalian beta defensin to an individual suffering from inflammatory bowel disease, comprising simultaneous or sequential oral and parenteral administration of said mammalian beta defensin.   98. The method of claim 97, wherein the mammalian beta defensin is administered orally after at least one parenteral administration of the drug.   98. The method of claim 97, wherein the mammalian beta defensin is administered orally with at least one parenteral administration of the drug dose.   99. The method of claims 97-99, wherein a defensin is administered orally and a defensin is administered parenterally, wherein the orally administered defensin and the parenterally administered defensin are the same. 99. The method of claims 97-99, wherein a defensin is administered orally and a defensin is administered parenterally, wherein the orally administered defensin and the defensin administered parenterally are different.

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